Measurement of Mouse and Human Interleukin 5

Gregory R. Harriman1

1 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.5
DOI:  10.1002/0471142735.im0605s14
Online Posting Date:  May, 2001
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Two fundamentally different types of assays are available for quantitating interleukin 5 in biological samples. One type, a bioassay, is based on the ability of IL‐5 to enhance B cell proliferation and immunoglobulin secretion or eosinophil proliferation and differentiation. The other type of assay, an ELISA, uses antibodies against IL‐5 to capture and quantitate IL‐5 in samples. Advantages and disadvantages of each assay are discussed. The bioassay described in this unit, utilizing BCL1 cells as the indicator line, has been designed primarily to assay murine IL‐5; however, it can also be used to measure human IL‐5. The IL‐5 ELISA, while sensitive and specific, provides no information about biological activity.

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Table of Contents

  • Basic Protocol 1: BCL1Proliferation Bioassay
  • Support Protocol 1: Maintenance of BCL1 Cells In Vivo
  • Basic Protocol 2: Enzyme‐Linked Immunosorbent Assay for IL‐5
  • Reagents and Solutions
  • Commentary
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Basic Protocol 1: BCL1Proliferation Bioassay

  • BCL 1 cells ( protocol 2)
  • Complete RPMI‐10 medium ( appendix 2A) with 15 mM HEPES
  • IL‐5 standard (Genzyme #MIL‐5)
  • [3H]thymidine ( appendix 3A)
  • 96‐well flat‐bottom microtiter plate (Costar)
  • Additional reagents and equipment for labeling and harvesting cells and determining [3H]thymidine incorporation ( appendix 3A)

Support Protocol 1: Maintenance of BCL1 Cells In Vivo

  Additional Materials
  • BALB/c mice (Jackson Lab or Charles River Labs)
  • Anti‐Thy‐1.2 antibody (ATCC #TIB 99 or #TIB 107)
  • Anti‐Lyt‐2 antibody (ATCC #TIB 105)
  • Additional reagents and equipment for mouse injection (unit 1.6), splenectomy (unit 1.10), preparation of cell suspensions (unit 3.1), T cell depletion (unit 3.4), macrophage removal by Sephadex G‐10 passage or plate adherence (units 3.6 & 3.7, respectively) and Ficoll‐Hypaque gradient centrifugation (unit 7.1), and 3.NaNcryopreservation of cells ( 3.NaN)

Basic Protocol 2: Enzyme‐Linked Immunosorbent Assay for IL‐5

  • TRFK‐4 and TRFK‐5 anti‐IL‐5 MAb (Dr. Robert Coffman, DNAX, Palo Alto, CA; unit 2.5)
  • Carbonate buffer ( appendix 2A)
  • 0.05% Tween 20 in phosphate‐buffered saline (Tween/PBS; appendix 2A)
  • 1% bovine serum albumin in PBS (BSA/PBS)
  • recipeBiotinylated TRFK‐4
  • Streptavidin–alkaline phosphatase (Southern Biotechnology #7100‐04)
  • recipeSubstrate solution
  • 96‐well ELISA plate (Corning #25805‐96)
  • Microtiter plate reader
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Literature Cited

Literature Cited
   Brooks, K., Yuan, D., Uhr, J.W., Krammer, P.H., and Vitetta, E.S. 1983. Lymphokine‐induced IgM secretion by clones of neoplastic B cells. Nature (Lond.) 302:825‐826.
   Campbell, H.D., Tucker, W.Q.J., Hort, Y., Martinson, M.E., Mayo, G., Clutterbuck, E.J., Sanderson, C.J., and Young, I.G. 1987. Molecular cloning, nucleotide sequence,and expression of the gene encoding human eosinophil differentiation factor (interleukin 5). Proc. Natl. Acad. Sci. U.S.A. 84:6629‐6633.
   Clutterbuck, E., Sheilds, J.G., Gordon, J., Smith, S.H., Boyd, A., Callard, R.E., Campbell, H.D., Young, I.G., and Sanderson, C.J. 1987. Recombinant human interleukin 5 is an eosinophil differentiation factor but has no activity in standard human B cell growth factor assays. Eur. J. Immunol. 17:1743‐1750.
   Coffman, R.L., Shrader, B., Carty, J., Mosmann, T.R., and Bond, M.W. 1987. A mouse T cell product that preferentially enhances IgA production. I. Biological characterization. J. Immunol. 139:3685‐3690.
   Murray, P.D., Swain, S.L., and Kagnoff, M.F. 1985. Regulation of the IgM and IgA anti‐dextran B1355S response: Synergy between IFN‐γ,BCGF II and IL‐2. J. Immunol. 135:4015‐4019.
   Murray, P.D., McKenzie, D.T., Swain, S.L., and Kagnoff, M.F. 1987. Interleukin 5 and interleukin 4 produced by Peyer's patch T cells selectively enhance immunoglobulin A expression. J. Immunol. 139:2669‐2674.
   Puré, E., Isakson, P.C., Takatsu, K., Hamaoka, T., Swain, S.L., Dutton, R.W., Dennert, G., Uhr, J.W., and Vitetta, E.S. 1981. Induction of B cell differentiation by T cell factors. I. Stimulation of IgM secretion by products of a T cell hybridoma and a T cell line. J. Immunol. 127:1953‐1958.
   Sanderson, C.J., Warren, D.J., and Strath, M. 1985. Identification of a lymphokine that stimulates eosinophil differentiation in vitro. Its relationship to interleukin 3,and functional properties of eosinophils produced in cultures. J. Exp. Med. 162:60‐74.
   Schumacher, J.H., O'Garra, A., Schrader, B., Van Kimmenade, A., Bond, M.W., Mosmann, T.R., and Coffman, R.L. 1988. The characterization of four monoclonal antibodies specific for mouse IL‐5 and development of mouse and human IL‐5 enzyme‐linked immunosorbent. J. Immunol. 141:1576‐1581.
   Swain, S.L. and Dutton, R.W. 1982. Production of a B cell growth‐promoting activity, (DL)BCGF, from a cloned T cell line and its assay on the BCL1 B cell tumor. J. Exp. Med. 156:1821‐1834.
   Takatsu, K., Tominaga, A., and Hamaoka, T. 1980. Antigen‐induced T cell‐replacing factor (TRF). I. Functional characterization of a TRF‐producing helper T cell subset and genetic studies on TRF production. J. Immunol. 124:2414‐2422.
   Takatsu, K., Kikuchi, Y., Takahashi, T., Honjo, T., Matsumoto, M., Harada, N., Yamaguchi, N. and Tominaga, T. 1987. Interleukin 5, a T‐cell‐derived B‐cell differentiation factor also induces cytotoxic T lymphocytes. Proc. Natl. Acad. Sci. U.S.A. 84:4234‐4238.
Key Reference
   Möller, G. (ed.) 1988. Interleukin 4 and interleukin 5. Immunol. Rev. 102 (entire issue).
  Contains review articles by most of the investigators who have been responsible for identifying and characterizing the properties of IL‐5.
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